A method for one-step freezing of mouse embryos

Effect of equilibration period on the viability of frozen‐thawed mouse morulae after rapid freezing

“…We have demonstrated that 30.2% of rapid frozen embryos (N = 86) developed to normal young when embryos were exposed to 2.0 M glycerol + 0.25 M lactose for 5 min (Takahashi and Kanagawa, 1985). Our previous result is similar to results reported for rapid freezing of mouse embryos (Miyamoto and Ishibashi, 1986;Scheffen et al, 1986;Williams and Johnson, 1986;Rall et al, 1987;Szell and Shelton, 1987;Trounson et al, 1988). Based on the results of our experiments, we recommend that embryos be placed in PBSCS containing 2.0 M glycerol and 0.25 M lactose for 3-5 min and that embryos be loaded in a 0.25 ml plastic straw during this exposure period, plunged into liquid nitrogen vapor by putting the straw on the Styrofoam boat floating on the liquid nitrogen bath, and then stored in liquid nitrogen.…”

“…The rapid freezing of embryos has usually been done by direct plunging into liquid nitrogen (Takeda et al, 1984;Krag et al, 1985;Biery et al, 1986;Trounson et al, 1987Reichenbach and Rodrigues, 1988) or into liquid nitrogen vapor Kanagawa, 1985, 1988;Shelton, 1986a,b, 1987;Williams and Johnson, 1986), but, during direct plunging into liquid nitrogen, straws are sometimes broken. We have used the cooling system described in the present text to prevent the breakage of straws and to keep the cooling rate constant Kanagawa, 1985, 1988).…”

“…The former freezing procedure requires only a brief, one-step exposure of embryos to the cryoprotectant solution at room temperature, followed by direct plunging into liquid nitrogen. Mouse embryos can be frozen in glycerol plus sucrose (Takeda et al, 1984;Biery et al, 1986;Williams and Johnson, 1986;Shelton, 1986a,b, 1987;Reichenbach and Rodrigues, 1988); trehalose (Krag et al, 1985); raffinose, lactose, or glucose (Takahashi and Kanagawa, 1985); dimethyl sulfoxide plus sucrose (Trounson et al, 1987, 19881; and 1, 2-propanediol plus sucrose (Sathananthan et al, 1988). Regarding the exposure to cryoprotectant solution, however, only Szell and Shelton (1986b) have demonstrated that the effect of equilibration at 20°C in glycerol-sucrose mixture was not significant, and these authors concluded that complete equilibration was not necessary.…”

Genome cryopreservation: a valuable contribution to mammalian genetic research

“…DMSO, propylene glycol (PROH), glycerol), at rates of cooling and warming ranging from < 1 to > 20Q0 °C/min. Also, the non-permeating property of sucrose has been used effectively to achieve some dehydration before freezing and to assist in the 'Whittingham et al (1972); 2 Wilson & Quinn (1989); 3 Trounson et al (1987); " Shaw & Trounson (1989); 5 Trounson et al (1988); 6 Bernard & Fuller (1983); ; 8 Miyamoto & Ishibashi (1983); 9 Massip et al (1984); 10 Szell & Shelton (1987); " Williams & Johnson (1986); 12 Reichenbach & Rodrigues (1988); 13 M. J. Wood & D. G. Whittingham (unpublished data); " Renard & Babinet (1984); 15 Miyamoto & Ishibashi (1978);16 Rall et al (1984); 17 Miyamoto & Ishibashi (1977).…”